Several publications are referenced in this application by numerals in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
In the current state of the art in the diagnosis and treatment of cancers and tumors, it is of critical importance that a clinician or pathologist be able to stage cancer progression in a patient. The more accurately the clinician or pathologist can stage a tumor or a cancer, the greater the chances that an efficacious, minimally toxic treatment can be devised. At the present time, however, there are few reliable diagnostic markers in clinical use for the staging of various types of tumors or cancers, particularly those having a high degree of prevalence.
In addition to the direct examination of a biopsy sample, clinicians and pathologists presently rely heavily upon the presence or absence of a number of genetic or protein markers in a sample obtained from a patient in order to stage a tumor. Diagnostic or prognostic indicators such as oncogenes, growth factors, tumor suppressors, tumor-associated proteases, the loss of heterozygosity at particular alleles, chromosomal aberrations, and the like are examples of those indicators or markers currently in routine use.
An example of such a protein marker is the cell adhesion protein E-cadherin. The loss of this protein in a tumor cell or tissue is a hallmark of many later stage tumor types (Siitonnen et al., 1996, Am. J. Clin. Path., 105:394–401). Late stage tumors are generally primed biochemically, though not necessarily triggered, to metastasize (i.e., to migrate from their site of origin and to potentially invade and colonize a distant site). Immunostaining for the presence or loss of this critical marker is widely used as a standard indicator of the stage of progression of a tumor. In particular, this marker is routinely used for staging epithelial tumors, such as breast (Dahiya et al., 1998, Breast Cancer Res. & Treat., 52:185–200), colon, stomach, esophagus, bladder, and liver tumors, as well as for staging cancers such as prostate cancer, melanoma, and squamous cell carcinomas of the head and neck.
Several other markers specific for particular tumor or cancer types have been used with increasing frequency over the last five years. BRCA1 and BRCA2 in the context of breast and ovarian cancer are examples of such markers (Dahiya et al., 1998, Breast Cancer Res. & Treat., 52:185–200).
The regulation of programmed cell death and survival plays a critical role in development, homeostasis, and malignant cell transformation. In addition to carcinogenesis (59), cellular survival programs are particularly important for regulation of normal lymphopoiesis (5) and the development of the nervous system (49). A number of pathways and factors that promote survival or antagonize apoptosis have been characterized. It is now well established that disruption of the balance between the members of the Bcl-2 family which are either pro-(e.g. Bax, Bad, Bak) or anti- (e.g. Bcl-2, BCL-XL, Bag) apoptotic, can ultimately affect the integrity of the mitochondrial membrane, resulting in the release of cytochrome C and activation of caspase enzymes, an ultimate and irreversible step in the apoptotic program (72). Members of the inhibitors of apoptosis protein (IAP) family of proteins suppress cell-death programs through their conserved baculoviral IAP repeats, by binding and inhibiting specific caspases (15). The pro-survival receptors of the TNFR family mediate their effects through the activation of MAPK/ERK cascades, resulting in the activation of the rel (NF-κB) and AP-1 transcription factor families which in turn transactivate genes of anti-apoptotic proteins, including IAPs (4, 16).
The transcriptional repressor ZEB (zfh-1/delta EF1) is a phylogenetically conserved DNA-binding protein containing eight kruppel-class zinc-finger domains as well as a homeodomain (21, 77, 74, 22, 24, 25). Identified based on its ability to bind E-box (CANNTG) motifs, ZEB has been implicated in the regulation of expression of a number of mammalian genes harboring such E-boxes. These include muscle-specific genes [the alpha-1 subunit of Na+, K+-ATPase (74), and muscle creatine kinase (63, 52)], genes specific for hematopoietic cells [interleukin 2 (77, 79), the immunoglobulin heavy chain (24), gata-3 (26), CD4 (9)], and other genes [delta-1 crystalline (22), alpha-4 integrin (31), ovalbumin (66), and E-cadherin (27)]. E-boxes, key regulatory elements in many promoters and enhancers, are known to bind the basic Helix-Loop-Helix (bHLH) class of trancriptional activators (41). Several bHLH transcriptional activators have been shown to trigger cell-type-specific differentiation programs (6, 41). The use of any of the above-identified molecules as markers for cancer progression has not yet been described.